Radiofluorination of a highly potent ATM inhibitor as a potential PET imaging agent

Purpose Ataxia telangiectasia mutated (ATM) is a key mediator of the DNA damage response, and several ATM inhibitors (ATMi) are currently undergoing early phase clinical trials for the treatment of cancer. A radiolabelled ATMi to determine drug pharmacokinetics could assist patient selection in a move towards more personalised medicine. The aim of this study was to synthesise and investigate the first 18F-labelled ATM inhibitor [18F]1 for non-invasive imaging of ATM protein and ATMi pharmacokinetics. Methods Radiofluorination of a confirmed selective ATM inhibitor (1) was achieved through substitution of a nitro-precursor with [18F]fluoride. Uptake of [18F]1 was assessed in vitro in H1299 lung cancer cells stably transfected with shRNA to reduce expression of ATM. Blocking studies using several non-radioactive ATM inhibitors assessed binding specificity to ATM. In vivo biodistribution studies were performed in wild-type and ATM-knockout C57BL/6 mice using PET/CT and ex vivo analysis. Uptake of [18F]1 in H1299 tumour xenografts was assessed in BALB/c nu/nu mice. Results Nitro-precursor 2 was synthesised with an overall yield of 12%. Radiofluorination of 2 achieved radiochemically pure [18F]1 in 80 ± 13 min with a radiochemical yield of 20 ± 13% (decay-corrected) and molar activities up to 79.5 GBq/μmol (n = 11). In vitro, cell-associated activity of [18F]1 increased over 1 h, and retention of [18F]1 dropped to 50% over 2 h. [18F]1 uptake did not correlate with ATM expression, but could be reduced significantly with an excess of known ATM inhibitors, demonstrating specific binding of [18F]1 to ATM. In vivo, fast hepatobiliary clearance was observed with tumour uptake ranging 0.13–0.90%ID/g after 1 h. Conclusion Here, we report the first radiofluorination of an ATM inhibitor and its in vitro and in vivo biological evaluations, revealing the benefits but also some limitations of 18F-labelled ATM inhibitors. Supplementary Information The online version contains supplementary material available at 10.1186/s13550-022-00920-z.


Chemical synthesis
All reagents used in the synthesis of compound 19 F-1 and 2 were purchased from commercial sources: Sigma Aldrich, Merck, Alfa Aesar, Fluorochem, Across Organics, Manchester Organics, Fisher Scientific.
Reactions were monitored characterised by silica thin layer chromatography (TLC), liquid chromatography mass spectrometry (LCMS, Waters HPLC and Acquity QDa Mass detection system), and NMR (400 MHz Bruker Ascend™). Novel compounds were characterised by 1 H, 13 C, 19 F NMR and high-resolution mass spectrometry (HRMS) on a Thermo Exactive High-Resolution Orbitrap FTMS using the HESI-II probe for positive electrospray ionization (ESI + ).

Automation of radiosynthesis
Automation

Western Blot full protocol
After the required treatments, cell culture medium was removed and cells were washed once with ice cold PBS. Cells were then lysed in RIPA buffer containing protease inhibitor (cOmplete EDTA-free,

H&E procedure
Frozen tissue section slides were allowed to air dry for 10 min at room temperature before washing in PBS (2 x 3 min) to remove any residual OCT. Slides were then submerged in 4% formaldehyde in PBS for 10 min to fix. Slides were washed in deionised water (3 x 2 min) before submerging in 0.1% Mayers Haematoxylin Solution (Sigma Aldrich) for 8 min at room temperature. Slides were then rinsed in cool running tap water for 6 min followed by rinsing in deionised water (2 x 2 min). Slides were dipped in Eosin Y-Solution 0.5% alcoholic (Merck) for 3.5 min then washed in distilled water for 2 min. Slides were dehydrated by dipping in ethanol solutions (70%, 90%, then 100%, 30 sec each) and cleared in xylene (30 sec) before mount and coverslip with DPX. Mounted slides were allowed to dry overnight before imaging at 20x on a Leica Aperio slide scanner.